On the relationship of OSW-1 to the cephalostatins

Antineoplastic bis-steroidal (cephalostatin-type) analogues of the saponin OSW-1 were produced from a dihydroaglycone of OSW-1. The key aglycone 6H was obtained from 5alpha-androstan-3beta-ol-17-one in 8 steps (38% yield). The SAR of the aglycones, intermediates, and hybrid analogues provide insights regarding the proposed common role of C22-oxocarbenium ions in the bioactivity of both OSW-1 and cephalostatins.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The &#103 -glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond &#103 -glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond &#103 -glucosidase. The substrate specificity of the almond &#103 -glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond &#103 -glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

Enzymatic Synthesis of Thioglucosides Using Almond β-glucosidase

A selection of different glycosidases was screened for the glycosylation of 1-propanethiol. The β-glucosidases from almond, Aspergillus niger and Caldocellum saccharolyticum were capable of 1-propanethioglucoside (1-PTG) formation. The almond β-glucosidase showed the highest activity in this reversed hydrolysis type of reaction using glucose as glucosyl donor. Besides 1-propanethiol, also thioglucosides of 2-propanethiol and furfuryl mercaptan were formed by the almond β-glucosidase. The substrate specificity of the almond β-glucosidase with respect to thioglucosylation is restricted to primary and secondary aliphatic thiols. Once the thioglucosides are formed, they are not hydrolyzed at a significant rate by almond β-glucosidase. As a consequence the synthesis of 1-PTG could be observed at very low aglycone concentrations (0.5% v/v based on the reaction solution) and high yields (68% based on 1-PT and 41% based on glucose) were obtained. An excess of aglycone, otherwise frequently applied in reversed hydrolysis glycosylation, is therefore not necessary in the glucosylation of 1-PT.     

Synthesis of alpha- and beta-glycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone

The synthesis of thioglycosyl donors with a disaccharide beta-D-Gal-(1-->3)-D-GalNAc backbone was studied using the glycosylation of a series of suitably protected 3-monohydroxy- and 3,4-dihydroxyderivatives of phenyl 2-azido-2-deoxy-1-thio-alpha- and 1-thio-beta-D-galactopyranosides by galactosyl bromide, fluoride, and trichloroacetimidate. In the reaction with the monohydroxylated glycosyl acceptor, the process of intermolecular transfer of thiophenyl group from the glycosyl acceptor onto the cation formed from the molecule of glycosyl donor dominated. When glycosylating 3,4-diol under the same conditions, the product of the thiophenyl group transfer dominated or the undesired (1-->4), rather than (1-->3)-linked, disaccharide product formed. The aglycone transfer was excluded when 4-nitrophenylthio group was substituted for phenylthio group in the galactosyl acceptor molecule. This led to the target disaccharide, 4-nitrophenyl 2-azido-4,5-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-beta-D-galactopyranoside, in 57% yield. This disaccharide product bears nonparticipating azide group in position 2 of galactosamine and can hence be used to form alpha-glycoside bond. 2-Azide group and the aglycone nitro group were simultaneously reduced in this product and then trichloroacetylated, which led to the beta-glycosyl donor, 4-trichloroacetamidophenyl 4,6-O-diacetyl-2-deoxy-3-O-(2,3,4,6-tetra- O-acetyl-beta-D-galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D- galactopyranoside, in 62% yield. The resulting glycosyl donor was used in the synthesis of tetrasaccharide asialo-GM1.     

Anticancer saponin OSW-1 is a novel class of selective Golgi stress inducer

OSW-1 is a plant-derived natural product proposed to selectively kill cancer cells by binding to members of the oxysterol binding protein family, thereby disrupting lipid/sterol homeostasis. However, how these protein-ligand interactions mediate cell death signaling has remained elusive. Here, we discovered that OSW-1 selectively activates the Golgi stress response leading to apoptosis, providing a mechanistic basis for the anticancer activity of OSW-1.     

Synthesis of OSW-1 analogs with modified side chains and their antitumor activities

Four analogs of OSW-1 (1-4) with modified side chains on the steroidal skeleton were synthesized following modification of our previous route for the total synthesis of OSW-1. Testing of the analogs against growth of tumor cells demonstrated that the 22-one function and the full length of the side chain of OSW-1 were not required for the antitumor action of OSW-1.     

Synthesis of a Highly Potent Antitumor Saponin OSW-1 and its Analogues

Twelve years ago a group of cholestane glycosides was isolated from the bulbs of Ornithogalum saundersiae, a species of the lily family without any medicinal folklore background. Similar glycosides were recently isolated from Galtonia candicans. The major component of the mixture of saponins, OSW-1, exhibited sub-nanomolar antineoplastic activity. While OSW-1 is exceptionally cytotoxic against various tumor cells, it shows little toxicity with normal human pulmonary cells. In this review article the synthetic efforts towards OSW-1 and related cholestane glycosides, as well as the preliminary results of the structure–activity relationship study are presented.     

Synthesis of biotinylated OSW-1

OSW-1 is a highly potent anticancer natural saponin with an unknown mode of action. To determine its cellular target(s) biotinylated OSW-1 was successfully synthesized in nine steps.     

Reaction of ascorbate with lysine and protein under autoxidizing conditions: formation of N epsilon-(carboxymethyl)lysine by reaction between lysine and products of autoxidation of ascorbate

N epsilon-(Carboxymethyl)lysine (CML) has been identified as a product of oxidation of glucose adducts to protein in vitro and has been detected in human tissue proteins and urine [Ahmed, M. U., Thorpe, S. R., & Baynes, J. W. (1986) J. Biol. Chem. 261, 4889-4894; Dunn, J. A., Patrick, J. S., Thorpe, S. R., & Baynes, J. W. (1989) Biochemistry 28, 9464-9468]. In the present study we show that CML is also formed in reactions between ascorbate and lysine residues in model compounds and protein in vitro. The formation of CML from ascorbate and lysine proceeds spontaneously at physiological pH and temperature under air. Kinetic studies indicate that oxidation of ascorbic acid to dehydroascorbate is required. Threose and N epsilon-threuloselysine, the Amadori adduct of threose to lysine, were identified in the ascorbate reaction mixtures, suggesting that CML was formed by oxidative cleavage of N epsilon-threuloselysine. Support for this mechanism was obtained by identifying CML as a product of reaction between threose and lysine and by analysis of the relative rates of formation of threuloselysine and CML in reactions of ascorbate or threose with lysine. The detection of CML as a product of reaction of ascorbate and threose with lysine suggests that other sugars, in addition to glucose, may be sources of CML in proteins in vivo. The proposed mechanism for formation of CML from ascorbate is an example of autoxidative glycosylation of protein and suggests that CML may also be an indicator of autoxidative glycosylation of proteins in vivo.     

GLYCOALKALOID METABOLISM1 is required for steroidal alkaloid glycosylation and prevention of phytotoxicity in tomato

Steroidal alkaloids (SAs) are triterpene-derived specialized metabolites found in members of the Solanaceae family that provide plants with a chemical barrier against a broad range of pathogens. Their biosynthesis involves the action of glycosyltransferases to form steroidal glycoalkaloids (SGAs). To elucidate the metabolism of SGAs in the Solanaceae family, we examined the tomato (Solanum lycopersicum) GLYCOALKALOID METABOLISM1 (GAME1) gene. Our findings imply that GAME1 is a galactosyltransferase, largely performing glycosylation of the aglycone tomatidine, resulting in SGA production in green tissues. Downregulation of GAME1 resulted in an almost 50% reduction in α-tomatine levels (the major SGA in tomato) and a large increase in its precursors (i.e., tomatidenol and tomatidine). Surprisingly, GAME1-silenced plants displayed growth retardation and severe morphological phenotypes that we suggest occur as a result of altered membrane sterol levels caused by the accumulation of the aglycone tomatidine. Together, these findings highlight the role of GAME1 in the glycosylation of SAs and in reducing the toxicity of SA metabolites to the plant cell.     

Excretion of miserotoxin and detoxification of the aglycone by grasshoppers (Orthoptera: Acrididae).

Two species of grasshoppers (Melanoplus bivittatus and M. sanguinipes) tolerated high levels of miserotoxin (3-nitro-1-propyl-beta-D-glucopyranoside) in their diet. Miserotoxin is a causative agent in cattle poisoning when timber milkvetch (Astragalus miser) is consumed. Toxic effects were averted by grasshoppers in part by excretion of the intact glycoside. When the aglycone was administered, detoxification was achieved by two routes: by oxidation of the aglycone to 3-nitropropionic acid which was then conjugated with glycine, and by glucosylation of the aglycone to miserotoxin, in each case followed by excretion.     

Synthesis and biological evaluation of alpha-galactosylceramide (KRN7000) and isoglobotrihexosylceramide (iGb3)

Glycoceramides can activate NKT cells by binding with CD1d to produce IFN-gamma, IL-4, and other cytokines. An efficient synthetic pathway for alpha-galactosylceramide (KRN7000) was established by coupling a protected galactose donor to a properly protected ceramide. During the investigation, it was discovered that when the ceramide was protected with benzyl groups, only beta-galactosylceramide was produced from the glycosylation reaction. In contrast, the ceramide with benzoyl protecting groups produced alpha-galactosylceramide. Isoglobotrihexosylceramide (iGb3) was prepared by glycosylation of Galalpha1-3Galbeta1-4Glc donor with 2-azido-sphingosine in high yield. Biological assays on the synthetic KRN7000 and iGb3 were performed using human and murine iNKT cell clones or hybridomas.     

Comparing the use of 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl and 2,4,6-trimethoxybenzyl as N-H protecting groups for p-tolyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-D-glucosides

A hurdle in glycosylation reactions of 2-acetamido glycosyl donors is the formation of a stable and unreactive oxazoline that decreases the yield of these reactions significantly. As an effort to prevent oxazoline formation during glycosylation reactions, we protected the N-H of the acetamido group within a 2-acetamido-2-deoxy-1-thio-β-D-glucoside with one of four different protecting groups. These groups were either 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl or 2,4,6-trimethoxybenzyl. The resulting N-alkylacetamides were then used in glycosylation reactions with ethanol as a model acceptor. We observed that the ethyl glycosides obtained in each case were obtained with exclusive β-selectivity without the formation of oxazoline sideproducts. The resulting products were then used to screen conditions for protecting group removal.     

Comparing the use of 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl and 2,4,6-trimethoxybenzyl as N–H protecting groups for p-tolyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-β-d-glucosides

A hurdle in glycosylation reactions of 2-acetamido glycosyl donors is the formation of a stable and unreactive oxazoline that decreases the yield of these reactions significantly. As an effort to prevent oxazoline formation during glycosylation reactions, we protected the N–H of the acetamido group within a 2-acetamido-2-deoxy-1-thio-β-d-glucoside with one of four different protecting groups. These groups were either 2-methylenenapthyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl or 2,4,6-trimethoxybenzyl. The resulting N-alkylacetamides were then used in glycosylation reactions with ethanol as a model acceptor. We observed that the ethyl glycosides obtained in each case were obtained with exclusive β-selectivity without the formation of oxazoline sideproducts. The resulting products were then used to screen conditions for protecting group removal.     

Cell-free translation of mouse liver mRNA coding for two forms of UDP glucuronosyltransferase

Mouse liver poly(A) RNA, when translated in vitro, produced two forms of UDP glucuronosyltransferase with molecular weights of approximately 50,000 and 54,000 (designated GTm1 and GTm2, respectively). These forms were identified by antibody prepared against GTm1. The mRNA coding for GTm1 was preferentially increased twofold after treatment of mice with 3-methylcholanthrene, while GTm2 mRNA was unaffected. Phenobarbital, however, increased the translatable levels of the mRNAs coding for both proteins approximately twofold. GTm1 was shown to be glycosylated during translation in the presence of dog pancreatic microsomes. This was reflected by a decrease in mobility of the protein in sodium dodecyl sulfate-polyacrylamide gels as compared to GTm1 translated in the absence of microsomes. Further evidence for glycosylation in vivo was demonstrated by an increase in the mobility of GTm1 immunoadsorbed from microsomes treated with endoglycosidase H. In contrast, GTm2 was unmodified. This apparent difference in the state of glycosylation may reflect a difference in the transmembrane distribution of these two enzyme forms, and hence play an important role in determining the type of aglycone glucuronidated and its route of removal from the cell.     

Oxidation of mitochondrial pyridine nucleotides by aglycone derivatives of adriamycin.

Adriamycin (AdM) and related anthracyclines are potent antineoplastic agents, the clinical utility of which is limited by severe cardiotoxicity. Aglycone derivatives of AdM have recently been reported to trigger the release of Ca2+ from isolated, preloaded rat heart mitochondria and to modify mitochondrial sulfhydryl (-SH) groups. Both mitochondrial Ca2+ retention and -SH status are sensitive to mitochondrial NAD(P)+/NAD(P)H ratios. This investigation examined the effects of AdM and its aglycone derivatives on the pyridine nucleotide redox status of isolated, intact heart mitochondria with the following results. (i) AdM aglycones induced the slow, Ca2(+)-independent oxidation of mitochondrial NAD(P)H. Oxidation was proportional to aglycone concentration between 5 and 60 microM. (ii) In terms of potency, 7-deoxy AdM aglycone greater than or equal to 7-hydroxy AdM aglycone much greater than AdM. (iii) Inhibitor data suggested that NAD(P)H oxidation reflects the rotenone-insensitive reduction of AdM aglycone and subsequent electron transfer to O2 generating superoxide. (iv) NAD(P)H oxidation mediated by AdM aglycone could be distinguished from the Ca2(+)-dependent NAD(P)H oxidation associated with mitochondrial Ca2+ release. This communication is the first to describe redox interactions of AdM with intact mitochondria.     

Isomerization of trifluoroacetamidopropyl-2-acetamido-2-deoxy-D- galactopyranoside into alpha-anomer. A convenient method of synthesis of the artificial T-antigen

Bioside Gal beta 1-3GalNAc alpha 1-O(CH2)3NHCOCF3 has been synthesized. The key alpha-glycoside GalNAc alpha 1-O(CH2)3NHCOCF3 (peracetate) was obtained either by isomerization of its beta-anomer with trifluoromethanesulfonic acid, or by direct glycosylation of 3-(trifluoroacetamido)propanol with D-galactosamine (anomeric pentaacetate) in the presence of a mixture of trifluoromethanesulfonic acid and boron trifluoride etherate. De-O-acetylated alpha-galactosaminide obtained was further transformed into benzylidene derivative, the latter was glycosylated with acetobromogalactose to give the protected alpha-bioside. The removal of the protecting groups gave the (3-aminopropyl)-alpha-bioside, which was subsequently immobilized on bovine serum albumin and cytochrome c.     

N-linked glycosylation status of classical swine fever virus strain Brescia E2 glycoprotein influences virulence in swine

E2 is one of the three envelope glycoproteins of classical swine fever virus (CSFV). Previous studies indicate that E2 is involved in several functions, including virus attachment and entry to target cells, production of antibodies, induction of protective immune response in swine, and virulence. Here, we have investigated the role of E2 glycosylation of the highly virulent CSFV strain Brescia in infection of the natural host. Seven putative glycosylation sites in E2 were modified by site-directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E2 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all putative glycosylation sites in E2 but restored when mutation N185A reverted to wild-type asparagine produced viable virus that was attenuated in swine. Single mutations of each of the E2 glycosylation sites showed that amino acid N116 (N1v virus) was responsible for BICv attenuation. N1v efficiently protected swine from challenge with virulent BICv at 3 and 28 days postinfection, suggesting that glycosylation of E2 could be modified for development of classical swine fever live attenuated vaccines.     

Somatic antigens of Pseudomonas aeruginosa. The structure of O-specific polysaccharide chains of the lipopolysaccharides from P. aeruginosa O1 (Lányi), O3 (Habs), O13 and O14 (Wokatsch), and the serologically related strain NCTC 8505

Lipopolysaccharides from Pseudomonas aeruginosa O1 (Lányi classification), O3 (Habs classification), O13 and O14 (Wokatsch classification), and strain NCTC 8505, which is also related to serogroup O3 (Habs), have structurally similar O-specific polysaccharide chains built up of tetrasaccharide repeating units involving L-rhamnose (Rha), 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-galacturonic acid (GalNAcA), and a di-N-acyl derivative of bacillosamine (BacN): 2,4-diacetamido-2,4,6-trideoxy-D-glucose or 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutyramido]-D-glucose. The latter derivative was obtained free by solvolysis with hydrogen fluoride of carboxyl-reduced Habs O3 polysaccharide, and was identified by 1H-nuclear magnetic resonance spectroscopy and by mass spectrometry of the corresponding methylated alditol. Habs O3, Lányi O1, and Wokatsch O14 polysaccharides contained O-acetyl groups. Solvolysis with hydrogen fluoride of the native Habs O3 polysaccharide resulted in selective cleavage of the glycosidic linkages of 6-deoxy sugars to give the trisaccharide fragment involving all three N-acylated amino sugars. Similar solvolysis of NCTC 8505 polysaccharide afforded a mixture of disaccharide and trisaccharide with N,N'-diacetylbacillosamine at the reducing end. Smith degradation of Habs O3 polysaccharide resulted in selective oxidation of rhamnose to give a glycoside of a trisaccharide with glyceraldehyde as the aglycone. Smith degradation of NCTC 8505 polysaccharide was complicated by the formation of the glycoside of a trisaccharide with an aglycone of unknown structure. A trisaccharide with rhamnose at the reducing end was also isolated after Smith degradation of the latter polysaccharide. Analysis of the composition and structure of all oligosaccharides obtained, and detailed examination of the 13C-nuclear magnetic resonance spectra of these oligosaccharides, and of both intact and modified polysaccharides, revealed the following structures of the repeating units. The structure for the NCTC 8505 polysaccharide differs from that proposed previously [Tahara, Y. and Wilkinson, S.G. (1983) Eur. J. Biochem. 134, 299-304] in the configurations assigned to the glycosidic linkages of rhamnose and bacillosamine. The results obtained show the P. aeruginosa strains studied to represent three different O-serotypes in a single O-serogroup (Formula: see text).